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Tytuł:
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Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity.
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Autorzy:
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Susini V; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.
Fierabracci V; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.
Barria G; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.
Dodoli L; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.
Caponi L; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.; Laboratory of Clinical Pathology, Santa Chiara University Hospital, Via Roma 67, 56126 Pisa, Italy.
Paolicchi A; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.; Laboratory of Clinical Pathology, Santa Chiara University Hospital, Via Roma 67, 56126 Pisa, Italy.
Franzini M; Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Via Savi 10, 56126 Pisa, Italy.; Laboratory of Clinical Pathology, Santa Chiara University Hospital, Via Roma 67, 56126 Pisa, Italy.
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Źródło:
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Biosensors [Biosensors (Basel)] 2021 Dec 03; Vol. 11 (12). Date of Electronic Publication: 2021 Dec 03.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Original Publication: Basel, Switzerland : MDPI Pub.
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MeSH Terms:
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Antigens*
Immunosorbents*
Antibodies ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Humans ; Male
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References:
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Contributed Indexing:
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Keywords: antibody avidity; half-fragment antibodies; immunoassay; oriented binding; reduced IgG; steric hindrance
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Substance Nomenclature:
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0 (Antibodies)
0 (Antigens)
0 (Epitopes)
0 (Immunosorbents)
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Entry Date(s):
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Date Created: 20211223 Date Completed: 20220310 Latest Revision: 20220310
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Update Code:
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20240104
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PubMed Central ID:
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PMC8699147
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DOI:
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10.3390/bios11120493
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PMID:
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34940250
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The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4-3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies.